This invention relates generally to the fields of protein synthesis and immunoassays and specifically relates to methods of synthesizing long chains of amino acids that contain multiple copies of epitopes for viruses such as HCV, and to assay devices that utilize multiple epitopes to detect the presence of antibodies.
In general, immunoassays are produced by first determining epitopes that are specifically associated with a virus and then determining which of the epitopes is preferred for the assay being developed. When the immunodominant epitopes are isolated, their sequences are determined, and genetic material for producing the immunodominant epitopes is produced. Methods of producing proteins by either chemical or biological means are known, as are assays used to detect the presence of antibodies to particular epitopes.
In producing immunoassays the overall object is to obtain an immunoassay which is both highly sensitive and highly selective. More specifically, the immunoassay must be designed such that it can detect even very low levels of the material it is designed to detect, i.e., it is highly sensitive. An assay having a high degree of sensitivity ensures that a sample, which has been tested, is not contaminated with the material the assay is designed to detect. For example, a highly sensitive assay that detects even the slightest presence of antibodies for a given virus is desirable in that it makes it possible to detect and thus discard samples that contain any amount of the antibody indicating that the samples contain the virus.
Although a high degree of sensitivity is desirable in an assay, it is not desirable if the assay is falsely indicating the presence of the material, i.e. the assay is providing a false positive result. Such false positive results can occur when the analyte has a high degree of similarity with another material present in the sample. The ability of an assay to differentiate between two similar but different materials relates to its selectivity.
An immunoassay with a high degree of selectivity will detect the presence of a material being assayed for even when that material is present in the sample in combination with other materials having a similar structure. Thus, a highly selective immunoassay will eliminate most false positive results. In general, as selectivity increases, sensitivity decreases. This occurs, in part, due to the high degree of variability in viruses. Thus, assays which are designed to be highly sensitive must take into account variability between different viruses. As virus variability is accommodated to improve sensitivity, the selectivity decreases. Alternatively, as one produces an immunoassay that is more and more selective with respect to a particular virus, the likelihood of the assay becoming so selective as to have decreased sensitivity, increases.
To a large extent, the problem of providing improved selectivity (less false positives) is dealt with by searching for and finding the most immunodominant epitopes. The problem of sensitivity (low concentration detection) is dealt with by providing immunodominant epitopes from a variety of different regions of the virus.
Current assays are designed to utilize relatively few peptides selected as xe2x80x9cmajor epitopesxe2x80x9d or highly immunodominant epitopes. Assay sensitivity is dependent on the number of major epitopes available on the solid support. If the availability of epitopes is limited by the number of peptides that can be coated on the solid phase, that assay will have reduced sensitivity. These results can be demonstrated as poor assay dilution sensitivity, poor seroconversion sensitivities and/or false negative determinations (Chien, D. Y. et al. (1993) J. Gastroent. Hepatol. 8:S33-39).
Accordingly, there is currently a need to improve the sensitivity and selectivity of assays for antibodies to pathogens in biological fluids and thereby improve diagnosis of pathogen infection resulting in improved screening of blood supplies.
Multiple copy fusion antigen (MEFA) immunoassays capable of detecting antibodies from multiple strains of a pathogen in a single assay are produced by (1) identifying nucleotide sequences that encode a plurality of different epitopes, including immunodominant components; (2) placing the nucleotide sequences into an expression cassette wherein at least two copies of a sequence coding for the same epitope region of an organism such as virus or corresponding regions of different strains of the virus is placed in a single cassette; (3) transforming a suitable host with one or more copies of the cassette in order to express sequences encoding epitopes, which sequences will include two or more copies of at least one epitope in a single chain antigen; (4) purifying the expressed multiple epitope antigen; and (5) adapting the purified multiple epitope antigen for an immunoassay, where adapting may include, but is not limited to, the following: coating the multiple epitope antigen on a surface of a substrate; covalently attaching a detectable marker to the multiple epitope antigen; and the like.
The purified epitopes are encompassed by the general structural formula (A)xxe2x80x94(B)yxe2x80x94(C)z which represents a linear amino acid sequence. B is an amino acid sequence of at least five and not more than 1,000 amino acids of an antigenic determinant or cluster of antigenic determinants, and y is an integer of 2 or more. Each copy of B is an equivalent antigenic determinant (for example, each copy is an epitope from a different viral strain). A and C are each independently an amino acid sequence of an epitope or cluster of epitopes not immediately adjacent to B in nature; and, x and z are each independently an integer of 0 or more, wherein at least one of x and z is 1 or more. Preferably the y epitopes of B are equivalent antigenic determinants from different viral strains thereby increasing the variety of pathogens detectable by a single multiple epitope antigen.
The selectivity is further improved by including immunodominant epitopes from the same region of two or more different strains of the same virus. More preferably, the equivalent antigenic determinants of B have different serotype specificity. Homology between the B epitopes is at least 30%, preferably at least 40%. The epitopes of the invention are more soluble, and are therefore more easily purified, than conventional epitopes. Further, the presence of repeating epitope sequences decreases masking problems and improves sensitivity in detecting antibodies by allowing a greater number of epitopes on a unit area of substrate. Sensitivity is further improved by placing the multiple copy epitopes of the invention on small spherical or irregularly shaped beads or microparticles thereby increasing the exposed surface area per given area of an assay device.
An object of the invention is to provide an amino acid sequence comprised of a plurality of epitopes wherein at least the antigenic determinant portion of at least one of the epitopes is repeated two or more times.
Another object of the invention is to provide a method of producing an immunoassay using multiple epitope fusion antigens.
A feature of the invention is that amino acid sequences that comprise multiple copies of a given epitope sequence have higher solubility as compared with amino acid sequences comprising only a single copy of any given epitope.
Another feature of the invention is that the nucleotide sequences encoding the epitopes are in a linear order that may be different from their linear order in the genome of the pathogen. Thus, the antigenic determinants of A, B, and C may be in a linear order different from the naturally occurring antigenic determinants of A, B and C. The linear order of the sequences of the invention is preferably arranged for optimum antigenicity of the expressed amino acid sequences comprising the multiple epitope fusion antigen.
An advantage of the invention is that the multi-epitope antigens of formula (I) can be more easily purified as compared with conventional epitopes.
Another advantage of the invention is that masking of an antigenic determinant can be reduced.
Another advantage of the invention is that the immunoassays utilizing the multiple epitope fusion antigens have improved sensitivity and selectivity.
Yet another advantage of the invention is that the multiple epitopes, particularly the repeated epitopes of B, provide an assay capable of detecting more than one pathogen or more than one strain of a single pathogen based on the type specificity of the epitopes.
Another feature of the invention is that the multiple epitope sequences of formula (I) can be designed to include a larger number and/or longer sequences than are generally present in epitope sequences containing only a single copy of any given epitope.
Another advantage of the invention is that the design of the multi-epitope antigens per formula (I) makes it possible to include a greater number of antigenic determinants on a unit area of surface of an immunoassay as compared to antigens containing only a single copy of any given epitope.
The invention also provides the advantage of improving the general specificity and sensitivity of serological tests when multiple epitopes are required and solid phase surface area is limiting. Additionally, immunoassay tests based on a single chimeric antigen will greatly simplify the manufacturing process, particularly for tests which require antigens labelled with detectable markers.
An embodiment of the invention further provides a rapid capture ligand immunoassay using multiple epitope fusion antigens that is simple and convenient to perform because it is a one step simultaneous assay. Detection is by the attachment of a detectable marker to a member of the antigen/antibody complex, preferably to the antigen. Attachment may be by covalent means or by subsequent binding of detectably labeled antibodies, such as a standard sandwich assay, or by enzyme reaction, the product of which reaction is detectable. The detectable marker may include, but is not limited to, a chromophore, an antibody, an antigen, an enzyme, an enzyme reactive compound whose cleavage product is detectable, rhodamine or rhodamine derivative, biotin, strepavidin, a fluorescent compound, a chemiluminescent compound, such as dimethyl acridinium ester (DMAE, Ciba Corning Diagnostics Corp.), derivatives and/or combinations of these markers.
In another embodiment of the invention, the capture ligand format assay contains a MEFA as an antigen, as well as an additional detectable epitope added to the assay mixture. The additional detectable epitope may be a single epitope or multiple epitopes and may include, but is not limited to, the epitopes included in the MEFA, preferably epitopes from regions such as E1, E2 and c33c. According to this embodiment of the invention, the additional epitope is attached or attachable to a detectable marker as described above. Where the additional epitope has preferred characteristics such as conformation, glycosylation, and the like, the additional epitope is expressed as a recombinant polypeptide from a cell, which expression provides the epitope in a desired form. Preferably, the epitope is obtainable from the cell using gentle isolation conditions that preserve the desired characteristics of the epitope. The cell may be any appropriate cell such as a mammalian cell, preferably a Chinese hamster ovary (CHO), or a bacterial, yeast or insect cell from which the additional epitope can be isolated in the desired form.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the multiple copy epitopes, immunoassays, and methods for producing such as more fully set forth below, with reference being made to the accompanying general structural formula forming a part hereof wherein like symbols refer to like molecular moieties throughout.